title: Atrial natriuretic peptide-mediated inhibition of microcirculatory endothelial Ca2+ and permeability response to histamine involves cGMP-dependent protein kinase I and TRPC6 channels.
abstract: OBJECTIVE
  Histamine increases microvascular endothelial leakage by activation of complex calcium-dependent and -independent signaling pathways. Atrial natriuretic peptide (ANP) via its cGMP-forming guanylyl cyclase-A (GC-A) receptor counteracts this response. Here, we characterized the molecular mechanisms underlying this interaction, especially the role of cGMP-dependent protein kinase I (cGKI).
  
  
  RESULTS
  We combined intravital microscopy studies of the mouse cremaster microcirculation with experiments in cultured microvascular human dermal endothelial cells. In wild-type mice, ANP had no direct effect on the extravasation of fluorescent dextran from postcapillary venules, but strongly reduced the histamine-provoked vascular leakage. This anti-inflammatory effect of ANP was abolished in mice with endothelial-restricted inactivation of GC-A or cGKI. Histamine-induced increases in endothelial [Ca(2+)]i in vitro and of vascular leakage in vivo were markedly attenuated by the Ca(2+)-entry inhibitor SKF96365 and in mice with ablated transient receptor potential canonical (TRPC) 6 channels. Conversely, direct activation of TRPC6 with hyperforin replicated the hyperpermeability responses to histamine. ANP, via cGKI, stimulated the inhibitory phosphorylation of TRPC6 at position Thr69 and prevented the hyperpermeability responses to hyperforin. Moreover, inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil prevented the edematic actions of histamine in wild types but not in mice with endothelial GC-A or cGKI deletion.
  
  
  CONCLUSIONS
  ANP attenuates the inflammatory actions of histamine via endothelial GC-A/cGMP/cGKI signaling and inhibitory phosphorylation of TRPC6 channels. The therapeutic potential of this novel regulatory pathway is indicated by the observation that sildenafil improves systemic endothelial barrier functions by enhancing the endothelial effects of endogenous ANP.
authors.affiliation: Institute of Physiology, University of Würzburg, Würzburg, Germany.
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.forename: Heike
authors.initials: H
authors.lastname: Oberwinkler
authors.forename: Franziska
authors.initials: F
authors.lastname: Werner
authors.forename: Birgit
authors.initials: B
authors.lastname: Gaßner
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Robert
authors.initials: R
authors.lastname: Feil
authors.forename: Franz
authors.initials: F
authors.lastname: Hofmann
authors.forename: Jens
authors.initials: J
authors.lastname: Schlossmann
authors.forename: Alexander
authors.initials: A
authors.lastname: Dietrich
authors.forename: Thomas
authors.initials: T
authors.lastname: Gudermann
authors.forename: Motohiro
authors.initials: M
authors.lastname: Nishida
authors.forename: Sabrina
authors.initials: S
authors.lastname: Del Galdo
authors.forename: Thomas
authors.initials: T
authors.lastname: Wieland
authors.forename: Michaela
authors.initials: M
authors.lastname: Kuhn
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002199:Capillary Permeability / Q000187:drug effects*; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D057809:HEK293 Cells; D006632:Histamine / Q000494:pharmacology*; D006633:Histamine Antagonists / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008297:Male; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D055806:Microvessels / Q000187:drug effects* / Q000201:enzymology; D058986:Phosphodiesterase 5 Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017461:Receptors, Atrial Natriuretic Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D050052:TRPC Cation Channels / Q000172:deficiency / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D000074287:TRPC6 Cation Channel; D013997:Time Factors; D014162:Transfection
chemical_list: D000893:Anti-Inflammatory Agents; D002121:Calcium Channel Blockers; D006633:Histamine Antagonists; D008565:Membrane Proteins; C485785:Mrvi1 protein, mouse; D058986:Phosphodiesterase 5 Inhibitors; D010750:Phosphoproteins; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C492146:Trpc6 protein, mouse; D006632:Histamine; D009320:Atrial Natriuretic Factor; D062965:Cyclic GMP-Dependent Protein Kinase Type I; C569718:PRKG1 protein, human; C569720:Prkg1 protein, mouse; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D002118:Calcium
keywords: TRPC6 channel; atrial natriuretic factor; cyclic GMP; cyclic GMP dependent protein kinase I; endothelial cells; guanylyl cyclase A